Compositions and methods for regulating RNA stability using polypyrimidine tract proteins

ABSTRACT

Compositions and methods for regulating CD154 gene expression are provided that rely on the interaction of polypyrimidine tract proteins with the 3′-untranslated region of CD154.

This application is a 371 of PCT/US03/01623 filed on Jan. 17, 2003,which claims benefit of 60/349,869 filed on Jan. 17, 2002 and claimsbenefit of 60/437,779 filed on Jan. 2, 2003.

INTRODUCTION

This invention was made in the course of research sponsored by theNational Institutes of Health (NIH Grant No. AI34928). The U.S.government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The expression of CD154 (CD40 ligand) by activated T lymphocytes iscritical in the development of humoral and cell-mediated immunity (Foy,et. al. (1996) Annu. Rev. Immunol. 14:591–617; Grewal and Flavell (1998)Ann. Rev. Immunol. 16:111–135; Noelle (1996) Immunity 4:415–419). Theinteraction of CD154 with its receptor, CD40, was first shown essentialfor B cell growth and differentiation and formation of germinal centers(Foy, et. al. (1996) Annu. Rev. Immunol. 14:591–617). This interactionis essential to numerous elements of cell-mediated immunity. In theabsence of CD154, antigen presentation by dendritic cells andmacrophages is profoundly impaired, as is macrophage-mediated killing ofintracellular or extracellular pathogens (Grewal and Flavell (1998)supra; Noelle (1996) supra). Given the breadth of importance ofCD154-CD40 interaction, it is not surprising that CD154 blockade retardsthe development and progression of immune responses in an array oftransplantation and autoimmune disease models ranging from SystemicLupus Erythematosus to Rheumatoid Arthritis to Multiple Sclerosis (Foy,et. al. (1996) supra; Grewal and Flavell (1998) supra).

The CD154 gene is located on the X chromosome, and belongs to the TumorNecrosis Factor (TNF) gene family (Hollenbaugh, et al. (1994) Immunol.Rev. 138:23–37). Study of CD154 expression chiefly involves CD4+ Tlymphocytes, with the earliest studies showing that resting cellsexpress little or no CD154 (Lane, et al. (1992) Eur. J. Immunol.22:2573–2578; Nusslein, et al. (1996) Eur. J. Immunol. 26:846–850; Roy,et al. (1993) J. Immunol. 151:2497–2510). Activation of the Tlymphocytes demonstrated that induction of CD154 expression wasdifferent from that of other cytokines. Signals (anti-CD3, mitogeniclectins) that triggered resting T cells to engage in high levels ofproliferation and cytokine production would elicit very little (CD4+ Tcells) or no (CD8+ T cells) expression on either mouse or human T cells(Lane, et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al.(1993) supra). Optimal expression of CD154 was found to requirepharmacologic stimulation provided by phorbol myristate acetate (PMA)and calcium ionophores such as ionomycin (Lane, et al. (1992) supra;Nusslein, et al. (1996) supra; Roy, et al. (1993) supra; Roy, et al.(1994) Eur. J. Immunol. 25:596–603). The induction of CD154 on Tlymphocytes is blocked by concurrent treatment with cyclosporine andglucocorticoids; these effects are presumed to be transcriptional(Fuleihan, et al. (1994) J. Clin. Invest. 93:1315–1320; Roy, et al.(1993) supra) based on the presence of NF-AT sites in the CD154 promoter(Schubert, et al. (1995) J. Biol. Chem. 15:29264–29627). Sincecyclosporine and glucocorticoids also inhibit cytokine production(Ashwell, et al. (1992) Ann. Rev. Immunol. 18:309–345; Sigal and Dumont(1992) Ann. Rev. Immunol. 10:519–60), this pathway does not account forthe differential regulation of CD154 expression by T lymphocytes.

The expression of TNF-α is primarily regulated at the level of mRNAturnover and translation, conferred by adenine-uridine rich cis-actingelements (AURE) present in its 3′-untranslated region (Beutler and Kruys(1995) J. Cardiovasc. Pharm. 25:S1–8; Kontoyiannis, et al. (1999)Immunity 10:387–398; Shaw and Kamen (1986) Cell 46:659–669). CD154 mRNAis rapidly degraded in human peripheral blood T lymphocytes, with ahalf-life of approximately 30 minutes, similar to that of interleukin 2(Ford, et al. (1999) J. Immunol. 162:4037–4044; Murakami, et al. (1999)J. Immunol. 163:2667–2673; Rigby, et al. (1999) J. Immunol.163:4199–4206; Suarez, et al. (1997) Eur. J. Immunol. 27:2822–2829).CD154 and cytokine mRNA stability may be differentially regulated inactivated T lymphocytes as evidenced by CD2 engagement by LFA-3stabilizes CD154 mRNA without altering IL-2 mRNA stability (Murakami, etal. (1999) supra) and CD28 crosslinking increases cytokine (TNF-α, IL-2)production at the level of mRNA stability (Lindsten, et al. (1989)Science 244:339–343) while having minimal effect on CD154 expression(Ford, et al. (1999) supra).

Using human peripheral blood lymphocytes (PBL) it was observed that PMAor ionomycin treatment rapidly increased CD154 mRNA stability, even inthe context of transcriptional inhibition (Rigby, et al. (1999) supra).In these studies, two major, p50 and p25, and two minor, p40 and p36,RNA binding proteins were shown to bind the CD154 3′-untranslatedregion. The binding of the p50 and p25 mapped to a polypyrimidine-richregion (˜0.4 kb) that lacked an AURE. UV crosslinking studiesdemonstrated that the p50 and p25 directly contacted uridines andcytidines in this region. Signals which stabilized CD154 mRNA decreasedp25 levels in both cytosolic and polysomal fractions, while acorresponding increase in p50 binding activity was observed (Rigby, etal. (1999) supra).

It has now been found that a novel cis-acting element in thispolypyrimidine-rich region exists and regulates CD154 mRNA turnoverthrough the relative levels of two polypyrimidine tract bindingproteins.

SUMMARY OF THE INVENTION

One aspect of the present invention is a polypyrimidine tract proteinisoform of SEQ ID NO:1.

Another aspect of the present invention is a method of increasing thestability of a ribonucleic acid operatively-linked to a cis-actingelement of a CD154 3′-untranslated region. The method providescontacting a cell or tissue containing a ribonucleic acid sequenceoperatively-linked to a cis-acting element of a CD154 3′-untranslatedregion with an agent which increases the level or activity of apolypyrimidine tract protein, such as polypyrimidine tract protein (PTB)of SEQ ID NO:2, which when bound to the cis-acting element of the CD1543′-untranslated region increases the stability of said ribonucleic acidsequence. The method further provides contacting the cell or tissue withan agent which decreases the level or activity of a polypyrimidine tractprotein, such as polypyrimidine tract protein isoform (PTB-T) of SEQ IDNO:1, which when bound to the cis-acting element of the CD1543′-untranslated region decreases the stability of said ribonucleic acidsequence. Increasing the level or activity of PTB or decreasing thelevel or activity of PTB-T increases the stability of a ribonucleic acidoperatively-linked to a cis-acting element of a CD154 3′-untranslatedregion in the cell or tissue. In a preferred embodiment, the cis-actingelement is SEQ ID NO:3.

A further aspect of the present invention is a method of decreasing thestability of a ribonucleic acid operatively-linked to a cis-actingelement of a CD154 3′-untranslated region. The method providescontacting a cell or tissue containing a ribonucleic acid sequenceoperatively-linked to a cis-acting element of a CD154 3′-untranslatedregion with an agent which decreases the level or activity of apolypyrimidine tract protein, such as polypyrimidine tract protein (PTB)of SEQ ID NO:2, which when bound to the cis-acting element of the CD1543′-untranslated region increases the stability of said ribonucleic acidsequence. The method further provides contacting the cell or tissue withan agent which increases the level or activity of a polypyrimidine tractprotein, such as polypyrimidine tract protein isoform (PTB-T) of SEQ IDNO:1, which when bound to the cis-acting element of the CD1543′-untranslated region decreases the stability of said ribonucleic acidsequence. Decreasing the level or activity of PTB or increasing thelevel or activity of PTB-T decreases the stability of a ribonucleic acidoperatively-linked to a cis-acting element of a CD154 3′-untranslatedregion in the cell or tissue. In a preferred embodiment, the cis-actingelement is SEQ ID NO:3.

A still further aspect of the invention is a method of preventing ortreating allograft rejection. The method provides administering to asubject in need of an allograft transplant an agent which decreases thelevel or activity of a polypyrimidine tract protein, such aspolypyrimidine tract protein (PTB) of SEQ ID NO:2, which when bound tothe cis-acting element of the CD154 3′-untranslated region increases thestability of the CD154 mRNA. The method further provides administeringto a subject in need of an allograft transplant an agent which increasesthe level or activity of a polypyrimidine tract protein, such aspolypyrimidine tract protein (PTB-T) of SEQ ID NO:1, which when bound tothe cis-acting element of the CD154 3′-untranslated region decreases thestability of the CD154 mRNA. Decreasing the level or activity of PTB orincreasing the level or activity of PTB-T decreases the stability ofCD154 mRNA thereby preventing or treating allograft rejection.

A further aspect of the invention is a method of inhibiting CD40activation. The method provides administering to a subject with adisorder associated with CD40 activation an effective amount of an agentwhich decreases the level or activity of a polypyrimidine tract protein,such as polypyrimidine tract protein (PTB) of SEQ ID NO:2, which whenbound to the cis-acting element of the CD154 3′-untranslated regionincreases the stability of the CD154 mRNA. The method further providesadministering to a subject with a disorder associated with CD40activation an effective amount of an agent which increases the level oractivity of a polypyrimidine tract protein, such as polypyrimidine tractprotein (PTB-T) of SEQ ID NO:1, which when bound to the cis-actingelement of the CD154 3′-untranslated region decreases the stability ofthe CD154 mRNA. Decreasing the level or activity of PTB or increasingthe level or activity of PTB-T decreases the stability of CD154 mRNAthereby inhibiting CD40 activation.

Another aspect of the invention provides a method of identifying anagent which modulates the level or activity of a polypyrimidine tractprotein. The method provides contacting a test cell containing acis-acting element of CD154 3′-untranslated region of SEQ ID NO:3operatively-linked to a nucleic acid sequence encoding a reporter, withan agent and detecting the expression of the reporter gene in the testcell in the absence and presence of said agent. Agents which decreasethe expression of the reporter are indicative of agents which decreasethe level or activity of PTB or increase the level or activity of PTB-T.Such agents are useful for preventing or treating allograft rejectionand inhibiting CD40 activation. Agents which decrease the expression ofthe reporter are indicative of agents which increase the level oractivity of PTB or decrease the level or activity of PTB-T.

These and other aspects of the present invention are set forth in moredetail in the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Two RNA binding proteins that bind to the 3′-untranslated region(3′-UTR) of the CD154 ligand gene have now been identified. These twobinding proteins are referred to as polypyrimidine tract bindingproteins as the sequence to which they bind is rich in polypyrimidine.One of these proteins was identified as polypyrimidine tract bindingprotein or PTB (SEQ ID NO:2), also known as hnRNP I, and has now beenshown to correspond to the cytoplasmic p50 CD154 3′-UTR binding protein.The other protein, which corresponds to p25, is a novel alternativelyspliced isoform of PTB and is known as PTB-T (SEQ ID NO:1). By bindingto polypyrimidine-rich sequences in the 3′-UTR of CD154, PTB-Tinfluences the levels of CD40 ligand that are produced. In contrast, PTBcompetes with PTB-T for binding sites in the 3′-UTR of CD40 ligand,regulating CD40 ligand expression at a post-transcriptional level.Regulation of the level or activity of PTB-T or PTB by pharmacologicstimuli is contemplated as a useful tool in the treatment of autoimmunediseases and allograft rejection.

Experiments were performed to characterize, purify and identifypolypyrimidine tract binding proteins as CD154 3′-UTR binding proteins.Excluding a ˜0.3 kb insert (nucleotides 1 to 292) immediately distal tothe translational stop site, the human and murine CD154 3′-UTR exhibit˜70% nucleotide identity between nucleotides 293 and 986. This level ofconservation is similar to that seen in the TNF-α 3′-UTR (67%), whichplays a dominant role in regulating its expression (Beutler and Kruys(1995) J. Cardiovasc. Pharm. 25:S1–8; Kontoyiannis, et al. (1999)supra). This conserved portion of the CD154 3′-UTR is distinguished bythe presence of CU-rich and polycytidine sequences, as well as a CAdinucleotide repeat. Though CD154 mRNA stability is comparable to IL-2(Rigby, et al. (1999) supra), it lacks the multiple AU-rich elements(AURE) seen in human TNF-α (9 AUUUA; SEQ ID NO:4) and IL-2 (7 AUUUA; SEQID NO:4) that occur within 500 nucleotides of the translational stopcodon. Rather, the conserved portion of the human CD154 3′-UTR has asingle distal AURE (UUAUUUAUU; SEQ ID NO:5) at nucleotides 951 to 959 ina context capable of destabilizing some, but not all, mRNA (Lagnado, etal. (1994) Mol. Cell. Biol. 14:7984–7995; Zubiaga, et al. (1995) Mol.Cell. Biol. 15:2219–2230).

Previous UV crosslinking studies identified RNA binding proteins with Mrof 50, 40, 36 and 25 kD in human peripheral blood lymphocyte (PBL)cytosols that directly contacted uridines and/or cytidines in theconserved region (nucleotides 293 to 986) of the human CD154 3′-UTR(Rigby, et al. (1999) supra). A similar pattern of CD154 3′-UTR bindingwas seen in ammonium sulfate (50%) precipitates of calf thymus (CT)nuclear extracts relative to cytosols from activated human PBL and theJurkat human T lymphocyte line. Each extract contained, to slightlyvarying degrees, the four major proteins previously shown to directlycontact radiolabeled [³²P]-UTP full-length CD154 3′-UTR. The p50, p40,and p25 binding activities mapped to the polypyrimidine-rich region(nucleotides 468 to 835) defined by the BstNI-HphI restriction enzymesites in the human CD154 3′-UTR cDNA. Deletion of nucleotides 483 to 814resulted in loss of p50, p40 and p25 binding in Jurkat cytosol and calfthymus extract. Activated human PBL cytosols also clearly demonstratereduced p25 binding activity with this RNA transcript, however thedecrease in p50 and p40 are obscured by the binding of additionalproteins of slightly different Mr. These results demonstrate that calfthymus nuclear extracts, Jurkat cells, and PBL exhibit identicalpatterns (p50, p40, p25) of binding to the polypyrimidine-rich portionof the human CD154 3′-UTR.

The p50 and p25 binding proteins were purified from the 50% ammoniumsulfate fraction of calf thymus nuclear extract by column chromatographyfor their ability to bind to radiolabeled nucleotides 468 to 835 in theCD154 3′-UTR. No significant binding to DEAE was noted; the flow throughwas applied to a carboxymethylcellulose (CMC) column. The p50 and p25binding activity eluted from the CMC column at 0.3–0.5 M NaCl, while thep40 binding activity was predominantly noted in the 0.1 M salt elution.The 0.3 M NaCl elution was subjected to polyuridine columnchromatography, where the p25 eluted at a slightly lower salt (0.5 M)concentration relative to the p50 (1 M NaCl). The 0.5 and 1 M NaClelutions were resolved by SDS-PAGE, with the p25 and p50 bands beingexcised after visualization with COOMASSIE® blue staining. The p50binding protein was identified by MALDI-TOF mass spectrometry aspolypyrimidine tract binding protein (PTB), also known as hnRNP I(Garcia-Blanco, et al. (1989) Genes Dev. 3:1874–1886; Ghetti, et al.(1992) Nucleic Acids Res. 20:3671–3678; Gil, et al. (1991) Genes Dev.5:1224–1236; Patton, et al. (1991) Genes Dev. 5:1237–1251). In contrast,the p25 could not be identified using this method or by N-terminalsequencing, the latter indicating that the amino terminus was blocked.Internal sequencing provided a nonapeptide(Asp-Tyr-Gly-Asn-Ser-Pro-Leu-His-Arg; SEQ ID NO:6) with 100% identity toamino acids 432 to 440 present in the third RNA Recognition Motif(RRM)-type RNA binding domain in human PTB (Garcia-Blanco, et al. (1989)supra; Patton, et al. (1991) supra).

Coordinate RNA binding assay and immunoblotting with the anti-PTBmonoclonal antibody BB7 (Chou, et al. (2000) Mol. Cell. 5:949–957)demonstrated reactivity of the p50 and p25 binding proteins in the crudecalf thymus nuclear extract as well as in the purified fractions. Aminor 40 kD doublet was also detected by immunoblotting in calf thymus,one isoform of which copurified with the p50. A similar correlation ofPTB-reactive proteins and CD154 3′-UTR binding activity was seen withJurkat cytosols as well as polyribosome-enriched (polysomes) fractionspurified by sucrose density gradients from PBL activated (6 hours) witheither PHA or PMA/ionomycin. PMA/ionomycin activation of PBL, whichinduces CD154 mRNA stabilization, is associated with a loss of p25binding activity, relative to PHA activation, from the polysomes whilethe p50 binding activity is increased and broadened (Rigby, et al.(1999) supra). Immunoblotting demonstrated that the changes in bindingactivity were associated with loss of the immunoreactive p25 PTBisoform, and increased levels of PTB as well as emergence of a second,slightly larger isoform. Polysomes from PHA-activated PBL, in whichCD154 mRNA is unstable, exhibited both p50 and p25 binding activity andPTB immunoreactivity.

Following incubation of Jurkat cytosol or calf thymus extract withradiolabeled CD154 3′-UTR RNA and UV-crosslinking, immunoprecipitationfor PTB and an irrelevant RNA binding protein (hnRNP A2) was performed.Anti-PTB antibody immunoprecipitated radiolabeled RNA-protein complexeswith a Mr of 50, 40, 25 kD from both Jurkat cytosol and calf thymusnuclear extract, indicating that each of these proteins directlycontacted the RNA and was related to PTB. Anti-hnRNP A2immunoprecipitated little (calf thymus) or no (Jurkat cytosol) bindingactivity, demonstrating the specificity of the observed data. These dataindicate that the p40 and p25 CD154 3′-UTR binding proteins are relatedto PTB. By the same criteria, the p36 binding activity, which exhibiteddifferent binding specificity, is unrelated to PTB.

The identification of the p50 and p25 as PTB or PTB-related proteins isconsistent with the description of multiple PTB splice isoforms andrelated, but distinct gene products. PTB was originally defined by itsregulation of alternative splicing. Full-length PTB consists of four RNArecognition motif (RRM)-type RNA binding domains, and homodimerizes dueto a region spanning the second RRM. Polypyrimidine tract bindingactivity is conferred by RRMs three and four (amino acids 324 to 531),particularly RRM three. Based on peptide sequence, binding activity,immunoreactivity and the inability to obtain N-terminal sequence, thedata indicated that the p25 represented an alternatively spliced isoformof PTB. Using either oligo-d(T) or PTB 3′-UTR-specific primers forreverse transcription followed by PCR with primers specific for 5′- and3′-UTR, RT-PCR amplification yielded a ˜700 bp product. The predicted(1700 bp) band corresponding to PTB was not well visualized under theseconditions, indicating preferential amplification of this smaller PCRproduct. This band was excised, cloned and sequenced, which confirmed itas a novel splice variant of PTB mRNA, with exons 3 though 9 deleted.The novel splice variant was confirmed by RT-PCR, cloning, andsequencing of three separate RNA samples, two from activated PBL fromdifferent donors, and one from the Jurkat T cell line. The splicingevent results in a deletion of 360 amino acids, over 50% of wild-typePTB, to produce a polypeptide corresponding in size to the p25. In vitrotranslation yielded a protein of comparable size to the observed bindingactivity. Furthermore, immunoblotting with antisera specific for theN-terminal 13 amino acids of PTB demonstrated equivalent reactivity ofthe p25 to that seen with the BB7 antibody in PBL cytosol. These dataprovide that the p25 is an alternatively spliced isoform of PTB,distinctly different the p25 form of PTB resulting from proteolysis(Bothwell, et al. (1991) J. Biol. Chem. 266:24657–63). Therefore, thep25 binding activity that correlated with mRNA turnover is encoded by anovel splice variant of PTB which retains most of RRM three and all ofRRM four, the domains that confer polypyrimidine tract binding activity.The PTB isoform was named PTB-T lymphocyte, or PTB-T, referring to thecell type in which it was first identified. However, expression analysisof PTB-T mRNA or protein indicates that PTB-T is not restricted tolymphoid cells.

Addition of PMA/ionomycin to PHA-activated human peripheral blood Tcells acutely increases CD154 mRNA stability, even in the context of RNApolymerase II inhibition. This effect is accompanied by decreased p25binding and increased p50 binding in the cytosol (Rigby, et al. (1999)supra). With the identification of PTB and PTB-T as alternately splicedisoforms that bind the CD154 3′-UTR, immunoblot analysis was conductedto investigate binding. Cytosolic levels of PTB and PTB-T were notsignificantly affected by short term (4 hours) activation by aconcentration of PHA that induced maximal proliferation and IL-2production. Addition of PMA/ionomycin rapidly increased PTB levels inconcert with a decline in PTB-T. RNA polymerase II inhibition by5,6-di-chloro-1-beta-D-ribofuranosylbenzimidazole (DRB) alone increasedcytoplasmic PTB. This effect was further enhanced by PMA/ionomycintreatment, indicating that the effect of PMA/ionomycin was independentof de novo gene transcription. Thus, cytosolic levels of PTB-T correlatewith an unstable CD154 mRNA. A corollary of these data is that while PHAactivation induces proliferation and lymphokine production by human PBL,it does not confer stabilization of CD154 mRNA. These data account forthe inability to detect CD154 expression in the absence of PMA/ionomycintreatment. This inability of PHA activation to alter the relativecytoplasmic levels of PTB-T/PTB was maintained for at least the first 16hours. With prolonged PHA activation (48 hours), a decline in cytosolicPTB-T was observed, consistent with reports that CD154 mRNA stabilityincreases with prolonged T cell activation.

Chimeric reporter gene constructs were used to analyze the role of CD1543′-UTR cis-acting elements in post-transcriptional gene regulation invivo to avoid non-specific toxicity and effects of transcriptionalinhibitors on mRNA stability. Firefly luciferase reporter constructspcDNA3.1/LUC and pcDNA3.1/LUC/CD154 104–986 were generated, in which theCMV immediate early promoter drives transcription of a luciferase mRNAlacking or containing the conserved portion of the human CD154 3′-UTR.In the Jurkat human T cell line, the presence of the CD154 3′-UTRreduced luciferase expression to 34% of that seen with cells transfectedwith identical reporter gene plasmids lacking this sequence. Acomparable level of inhibition of luciferase activity was conferred bythe CD154 3′-UTR in transient transfection of HeLa cells. In each celltype, the level of inhibition of luciferase activity conferred by theCD154 3′-UTR was statistically significant (p<0.001). The level ofinhibition of luciferase activity was equivalent to that seen withluciferase reporter constructs containing six reiterated AUUUA (SEQ IDNO:4) pentamers in their 3′-UTR. Thus, the CD154 3′-UTR containssequences that modulate luciferase reporter gene expression in apromoter-independent manner to the same extent as an AURE.

CD154 3′-UTR-regulated luciferase reporter gene expression intransiently transfected Jurkat T lymphocytes was examined. Followingtransfection with the pcDNA3.1/LUC and pcDNA3.1/LUC/CD154 104–986expression vectors, total cellular RNA was extracted and analyzed bynorthern blot. The presence of the CD154 3′-UTR reduced accumulation ofluciferase mRNA by 60%. This effect was comparable to the magnitude(50%) of the CD154 3′-UTR-dependent reduction in luciferase activitymeasured at the same time as the RNA extraction. These data demonstrateda direct relationship between mRNA accumulation and luciferaseexpression. In five separate experiments, the effect of the CD154 3′-UTRon luciferase mRNA accumulation was measured by real time quantitativeRT-PCR. The level of CD154 3′-UTR-dependent reduction in luciferase mRNAaccumulation was comparable to that seen by northern blot analysis.Thus, equivalent patterns of CD154 3′-UTR-dependent changes inluciferase mRNA accumulation were shown using distinct techniques. Theparallel between the magnitude of the effect of the CD154 3′-UTR onluciferase mRNA and luciferase activity indicates a change in mRNAstability, since the rate of transcription from each promoter should beequivalent. This is consistent with reports demonstrating theinstability of CD154 mRNA (Ford, et al. (1999) supra; Murakami, et al.(1999) supra; Rigby, et al. (1999) supra; Suarez, et al. (1997) supra)and the role of the 3′-UTR in regulating mRNA turnover (Ross (1988) Mol.Biol. Med. 5:1–14).

Deletion analysis was performed to map the cis-acting element in theCD154 3′-UTR. Removal of the entire polypyrimidine-rich region in the485–814 deletion construct resulted in a loss of inhibition ofluciferase activity. Constructs with smaller deletions of thepolypyrimidine-rich region all demonstrated inhibitory activity.Deletion of the polycytidine sequence, CU dinucleotide repeat-richregion (nucleotides 560 to 600) or even deletion of >75% of the CU-richregion (nucleotides 540 to 690) with the 468–549 deletion, 557–647deletion, or 585–690 deletion reporter constructs, still resulted inreduced luciferase expression in a 3′-UTR-dependent manner. Finally, thepolypyrimidine-rich region alone (nucleotides 474 to 835) of human CD1543′-UTR reduced luciferase activity to a comparable degree to that seenwith nucleotides 104 to 986. These effects were statisticallysignificant (p<0.0005).

When analyzed by transient transfection of Jurkat cells, both the CD1543′-UTR and the polypyrimidine-rich region alone (474 to 835) werecapable at reducing mRNA accumulation; deletion of this region resultedin loss of inhibition. Thus, the polypyrimidine-rich region of humanCD154 3′-UTR is both necessary and sufficient to reduce both luciferaseactivity and steady state luciferase mRNA accumulation in a3′-UTR-dependent manner. Importantly, this region (nucleotides 474 to835) lacks an AURE, indicating that this effect on reporter gene mRNAlevels is mediated by a cis-acting element.

A tetracycline-responsive luciferase (pTRE-luc) vector that eitherlacked or contained the CD154 3′-UTR was generated to conduct mRNAstability experiments in the absence of RNA polymerase II inhibitors.HeLa cells (TET-OFF™) were transiently transfected with these constructsand transcription was controlled with doxycycline. mRNA stability wasevaluated by quantitative RT-PCR analysis. The presence of the CD1543′-UTR resulted in a greater than two-fold increase in the rate ofluciferase mRNA decay. This effect of the CD154 3′-UTR required thepresence of the polypyrimidine-rich region; deletion of nucleotides 485to 814 exhibited increased mRNA stability relative to the CD154 3′-UTRvector. Interestingly, the 485 to 814 deletion construct demonstratedincreased mRNA stability relative to the control vector alone in twoexperiments. This finding is consistent with augmented mRNA accumulationrelative to the luciferase control seen with transient transfection ofJurkat T cells. Thus, the retained AURE (UUAUUUAUU; SEQ ID NO:5) in the485 to 814 deletion construct had no inhibitory effect on mRNAaccumulation, indicating it lacked activity in this context.

Overexpression of PTB or PTB-T in a transient transfection assay wasperformed to determine the differential regulation of CD1543′-UTR-dependent gene expression. In the Jurkat human T cell line,transfection of an expression vector encoding PTB increased CD1543′-UTR-dependent gene expression relative to empty vector controls. Incontrast, transfection of an expression vector encoding PTB-T markedlyreduced luciferase activity in a CD154 3′-UTR-dependent manner. Theseeffects were statistically significant. A similar, statisticallysignificant effect of PTB-T overexpression was seen in HeLa cells. Thesedata are consistent with the interpretation that levels of cytoplasmicPTB-T were limiting in both cell types. No effect of PTB overexpressionwas seen in HeLa cells, indicating the possibility that PTB levels inthese cells were not limiting. The presence of the polypyrimidine-richregion in the luciferase 3′-UTR was both necessary and sufficient toconfer the inhibitory effect of PTB-T transfection on luciferaseexpression.

A similar pattern was seen in transient transfection of purified humanCD4+ T lymphocytes. Following transfection, luciferase activity in CD4+T cells was measured after 6 hours either without (basal) or withPMA/ionomycin stimulation. The presence of the CD154 3′-UTR reducedluciferase activity in both HeLa and Jurkat cell lines under both basaland stimulated conditions. Moreover, in primary human CD4+ T cells, PTBand PTB-T overexpression differentially affected luciferase expression,in a CD154 3′-UTR-dependent manner. When expressed as a function oftheir effect on CD154 3′-UTR-dependent gene expression from fourexperiments, the inhibitory effect of PTB-T was statisticallysignificant. The selectivity of the effect of PTB-T transfection for theCD154 3′-UTR was demonstrable through its lack of effect on reportergene activity derived from the pcDNA3.1/LUC control. Furthermore, noeffect on pRL-null-derived Renilla luciferase activity was seen, whichwas used to control for transfection efficiency in each experiment. Inseparate experiments, it was demonstrated that, as in Jurkat cells, theeffect of PTB-T on luciferase expression in normal human CD4+ Tlymphocytes was conferred by nucleotides 468 to 835. PTB-T reducedluciferase expression by pcDNA3.1/LUC/CD154 104–986 by 38% relative tocontrols. Transfection of PTB-T cDNA had a greater effect (58%inhibition) of luciferase expression by pcDNA3.1/LUC/CD154 468–835(n=4). Together, these data show that PTB-T, and in some instances, PTB,regulated CD154 gene expression in both normal human T cells and celllines solely through their interaction with the polypyrimidine-richregion found in the 3′-UTR.

As demonstrated herein, the CD154 3′-UTR contains a cis-acting elementwhich decreases reporter gene expression at the level of mRNAaccumulation in vivo. The polypyrimidine-rich region of CD154 3′-UTR wassufficient to reduce reporter gene expression indicating the presence ofa cis-acting element in this region that regulates mRNA turnover. Thissame region also binds both PTB (SEQ ID NO:2) and a novel splice isoformnow identified as PTB-T (SEQ ID NO:1), which both function astrans-acting factors to regulate the function of the cis-acting element.Therefore, both PTB and PTB-T, by binding to the polypyrimidine-richregion of human CD154 3′-UTR, play critical roles in regulating CD154expression in vivo. Furthermore, it is believed that as cytoplasmiclevels of PTB increase, PTB-T is displaced due to the higher avidity ofdimeric PTB for binding to the CD154 3′-UTR. In contrast to PTB, PTB-Tlacks the homodimerization domain that encompasses RRM two. Dimeric PTBmay interact with the polypyrimidine-rich region of the CD154 3′-UTR atmultiple sites and have a distinct effect on RNA structure relative toPTB-T, thus favoring CD154 mRNA stability. Therefore, a novel pathwaythat involves PTB and PTB-T has been identified that results inpost-translational regulation of CD154 gene expression.

Accordingly, the present invention provides methods of modulating orregulating, in a cell or tissue, the stability of a ribonucleic acid(RNA) sequence operatively-linked to a cis-acting element of a CD1543′-UTR via the binding of a polypyrimidine tract protein such as PTB,PTB-T or isoforms thereof (e.g., p40). These methods provide contactingthe cell or tissue with an agent which modulates, regulates or altersthe level or activity of a polypyrimidine tract protein therebymodulating or regulating the stability of an RNA sequenceoperatively-linked to a cis-acting of a CD154 3′-UTR. For illustrativepurposes, PTB (SEQ ID NO:2) and PTB-T (SEQ ID NO:1) are used in thedisclosure of the present of the invention, however, it should beunderstood that polypyrimidine tract proteins which have bindingcharacteristics similar to PTB or PTB-T, e.g., p40, are alsocontemplated.

Methods of modulating or regulating the stability of an RNA sequenceoperatively-linked to a cis-acting element of a CD154 3′-UTR encompassboth increasing and decreasing the stability of said RNA sequence. Inone aspect of the invention, the stability of said RNA is increased bycontacting the cell or tissue with an agent which increases orstimulates the level or activity of PTB (SEQ ID NO:2) or decreases orinhibits the level or activity of PTB-T (SEQ ID NO:1). In another aspectof the invention, the stability of said RNA is decreased by contacting acell or tissue with an agent which decreases or inhibits the level oractivity of PTB (SEQ ID NO:2) or increases or stimulates the level oractivity of PTB-T (SEQ ID NO:1). The stability of said RNA by maydetermined using standard techniques such as western blot analysis ofthe translated product of the RNA sequence, northern blot analysis,reverse-transcriptase PCR, or other well-known methods for measuring RNAtranscript levels. Regulation of the level or activity of PTB-T or PTBby pharmacological agents is contemplated as a useful tool in thetreatment of autoimmune diseases and allograft rejection.

Accordingly, another aspect of the invention is a method of preventingor treating allograft rejection in a subject or mammalian recipient of atissue graft or any mammal in need of a tissue graft. The methodprovides decreasing the stability of CD154 mRNA prior to, during, and/orafter a tissue graft. The stability of the CD154 mRNA is decreased byadministering to said subject an effective amount of an agent whichdecreases or inhibits the level or activity of PTB (SEQ ID NO:2) orincreases or inhibits the level or activity of PTB-T (SEQ ID NO:1).Preferably, the subject is a primate, more preferably a higher primate,most preferably a human. In other embodiments, the subject may beanother mammal in need of a tissue graft, particularly a mammal ofcommercial importance, or a companion animal or other animal of value.Thus, subjects also include, but are not limited to, sheep, horses,cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, ratsand mice.

It is contemplated that the agent may be administered as a capsule,intramuscularly, intraperitoneally, subcutaneously, intradermally orapplied locally to a wound site. It is also clear that the invention canbe used with a skin graft procedure. The skin is a notoriously difficulttissue with which to achieve or maintain engraftment. A preferred routeof administration for treating or preventing skin graft rejection istopical, subdermal, intradermal or subcutaneous, though systemic andother routes are also contemplated.

Another preferred route of administration includes direct applicationlocally (by topical application, immersion or bath, or local injection)into the subject tissue bed, or to the graft tissue itself. High localconcentrations of the agent, particularly in areas of lymphaticdrainage, are expected to be particularly advantageous. Alternatively,the graft tissue may be transfected or transformed with a recombinantexpression vector to overexpress PTB-T or inhibit the expression of PTBby antisense expression of PTB.

An effective amount of an agent which decreases the level or activity ofPTB or increases the level or activity of PTB-T is an amount whichdecreases or inhibits the signs or symptoms of allograft rejection(e.g., edema, fever, and loss of graft function) and will be dependenton the nature of the agent. For example, an effective, non-toxic amountof an anti-PTB antagonistic antibody would generally be in the range ofabout 0.05 to 100 milligrams per kilogram body weight per day.

The method may be used with any type of tissue transplant or graftprocedure, particularly procedures wherein the donor (grafted) tissue isaffected by, or at risk of, failure or rejection by the subject's immunesystem. The donor tissue may be derived, by conventional means, from avolunteer or other living donor, or from a cadaveric donor. The donortissue may also be artificial tissue, such as artificial skin products.Preferably, the donor is as histocompatible as practicable with therecipient host.

The donor tissue comprises an organ, a portion of an organ, such as aliver, a kidney or a heart, or a body part comprising multiple tissuetypes such as a joint, a hand, a foot, a myocutaneous flap or a finger.The donor tissue may further comprise a part, portion or biopsy of adonor organ or tissue; isolated or suspended cells, including cellswithdrawn or excised from a donor host, cells maintained in primaryculture, or an immortalized cell line; cells harboring exogenous geneticmaterial, such as transfected or transformed host cells which have been(or are derived from ancestor cells which have been) engineered toinclude genetic material necessary for the production of a polypeptideof therapeutic value to the recipient host.

A further aspect of the invention is a method of inhibiting CD40activation. The method provides decreasing the stability of CD154 mRNAin a subject suffering from a disorder associated with CD40 activation.The stability of the CD154 mRNA is decreased by administering to saidsubject an effective amount of an agent which decreases or inhibits thelevel or activity of PTB (SEQ ID NO:2) or increases or inhibits thelevel or activity of PTB-T (SEQ ID NO:1). An effective amount of anagent which decreases the level or activity of PTB or increases thelevel or activity of PTB-T is an amount which decreases or inhibits thesigns or symptoms of CD40 activation (e.g., inflammation; renaldisorder; or B cell, macrophage, or dendritic cell activation) and willbe dependent on the nature of the agent. The subject may be a non-humanor, preferably, a human animal. Disorders associated with CD40activation include, but are not limited to, allergy (includinganaphylaxis); atherosclerosis; autoimmune conditions including druginduced lupus, systemic lupus erythematosus, adult rheumatoid arthritis,juvenile rheumatoid arthritis, scleroderma, Sjogren's Syndrome, etc.;and viral diseases that involve B-cells, including Epstein-Barrinfection, and retroviral infection including infection with a humanimmunodeficiency virus.

Because it has been suggested that B cell activation is associated withthe induction of human immunodeficiency virus replication from latency,it may be desirable to decrease the stability of CD154 mRNA in HIVpositive individuals who have not yet developed AIDS or ARC.

Agents useful in accordance with the methods provided herein include,but are not limited to, purified PTB or PTB-T protein, a recombinantexpression vector expressing PTB or PTB-T, a recombinant expressionvector expressing antisense PTB or PTB-T, antisense oligonucleotides toPTB or PTB-T, organic molecules, biomolecules including peptides,antibodies, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

An isolated or purified PTB or PTB-T protein for administration to acell or tissue may be produced by various means. An isolated or purifiedprotein is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the PTBor PTB-T protein is derived. To be substantially free of cellularmaterial includes preparations of PTB or PTB-T protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. When the PTB or PTB-T protein isrecombinantly produced, it is also preferably substantially free ofculture medium.

Recombinant production of PTB or PTB-T typically involves generating afusion protein such as a GST-PTB or GST-PTB-T fusion protein in whichthe PTB or PTB-T sequences are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant PTB or PTB-T. Alternatively, the fusion protein is a PTB orPTB-T protein containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of PTB or PTB-T can be increased through useof a heterologous signal sequence. Preferably, a PTB or PTB-T chimericor fusion protein of the invention is produced by standard recombinantDNA techniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques, for example by employing blunt-ended orstagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. Alternatively, the fusion gene may besynthesized by conventional techniques including automated DNAsynthesizers or PCR amplification. PCR amplification of gene fragmentsmay be carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which are subsequentlyannealed and reamplified to generate a chimeric gene sequence (see,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al. JohnWiley & Sons, 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).A PTB- or PTB-T-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thePTB or PTB-T protein.

A recombinant expression vector comprises a nucleic acid sequenceencoding PTB or PTB-T in a form suitable for expression of the nucleicacid sequence in a host cell, which means that the recombinantexpression vector includes one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which isoperatively-linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, operatively-linked is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell). A regulatory sequence is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel (1990) Methods Enzymol. 185:3–7. Regulatory sequences includethose which direct constitutive expression of a nucleic acid sequence inmany types of host cells and those which direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences). It will be appreciated by one of skill in the artthat the design of the expression vector depends on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, and the like. The expression vector may be introducedinto a host cell to thereby produce proteins or peptides of PTB orPTB-T, isoforms of PTB or PTB-T, mutant forms of PTB or PTB-T proteins,fusion proteins, and the like.

A recombinant expression vector may be designed for expression of PTB orPTB-T proteins in prokaryotic or eukaryotic cells. For example, PTB orPTB-T proteins may be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors), yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel(1990) supra. Alternatively, the recombinant expression vector may betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve to increase expression of recombinant protein;increase the solubility of the recombinant protein; and aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson (1988) Gene 67:31–40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann, et al., (1988) Gene 69:301–315) and pET 11d(Studier, et al. (1990) Methods Enzymol. 185:60–89). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3)or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene underthe transcriptional control of the lacUV 5 promoter.

The PTB or PTB-T expression vector may also encompass a yeast expressionvector. Examples of vectors for expression in yeast Saccharomycescerevisiae include pYepSec 1 (Baldari, et al. (1987) EMBO J. 6:229–234),pMFa (Kurjan and Herskowitz (1982) Cell 30:933–943), pJRY88 (Schultz, etal. (1987) Gene 54:113–123), pYES2 (INVITROGEN™ Corp., San Diego,Calif.), and picZ (INVITROGEN™ Corp., San Diego, Calif.).

Alternatively, PTB or PTB-T proteins may be expressed in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith, et al. (1983) Mol. Cell Biol.3:2156–2165) and the pVL series (Lucklow and Summers (1989) Virology170:31–39).

Further, nucleic acid sequences encoding PTB or PTB-T are expressed inmammalian cells using a mammalian expression vector. As will beappreciated by one of skill in the art, PTB or PTB-T expression inmammalian cells provides a means of purifying the proteins as well as ameans of modulating the endogenous levels of PTB or PTB-T proteins in acell. Examples of mammalian expression vectors include any one of thewell-known recombinant viral vectors, pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman, et al. (1987) EMBO J. 6:187–195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, etal. Molecular Cloning: A Laboratory Manual. 2^(nd) ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

The recombinant mammalian expression vector may further be capable ofdirecting expression of the nucleic acid preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe nucleic acid). Tissue-specific regulatory elements are known in theart. Non-limiting examples of suitable tissue-specific promoters includethe albumin promoter (liver-specific; Pinkert, et al. (1987) Genes Dev.1:268–277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235–275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729–733) and immunoglobulins(Banerji, et al. (1983) Cell 33:729–740; Queen and Baltimore (1983) Cell33:741–748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473–5477), pancreas-specific promoters (Edlund, et al. (1985)Science 230:912–916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and EP 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374–379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537–546).

In addition to increasing the expression of PTB or PTB-T to modulate thelevels of PTB or PTB-T present in the cell, PTB or PTB-T expression maybe decreased to modulate the levels of PTB or PTB-T present in the cell.Thus, a recombinant expression vector harboring a nucleic acid sequenceencoding PTB or PTB-T cloned into the expression vector in an antisenseorientation is also provided. That is, the nucleic acid sequenceencoding PTB or PTB-T is operatively-linked to a regulatory sequence ina manner which allows for expression (by transcription of the nucleicacid sequence) of an RNA molecule which is antisense to PTB or PTB-TmRNA. Regulatory sequences operatively-linked to a nucleic acid clonedin the antisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue-specific or celltype-specific expression of antisense RNA. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus in which antisense nucleic acids are produced under thecontrol of a high efficiency regulatory region, the activity of whichcan be determined by the cell type into which the vector is introduced.For a discussion of the regulation of gene expression using antisensegenes see Weintraub, et al. (1986) Reviews-Trends in Genetics Vol. 1(1).

Host cells into which a PTB or PTB-T nucleic acid sequence may beintroduced, e.g., a PTB or PTB-T nucleic acid sequence within a vector(e.g., a recombinant expression vector) or a PTB or PTB-T nucleic acidsequence containing sequences which allow it to homologously recombinedinto a specific site of the host cell's genome, are furthercontemplated. The terms host cell and recombinant host cell are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell may be any prokaryotic or eukaryotic cell. For example, aPTB or PTB-T protein may be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells areexemplified herein and are known to those skilled in the art.

Vector DNA may be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms transformation and transfection are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2^(nd), ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the nucleic acid sequence ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a PTB or PTB-T protein or may beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid may be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die). A host cell, such as a prokaryotic or eukaryotichost cell in culture, may be used to produce (i.e., express) a PTB orPTB-T protein.

The host cells may also be used to produce non-human transgenic animals.For example, a host cell is a fertilized oocyte or an embryonic stemcell into which PTB or PTB-T-coding sequences have been introduced. Suchhost cells may then be used to create non-human transgenic animals inwhich exogenous PTB or PTB-T sequences have been introduced into theirgenome or homologous recombinant animals in which endogenous PTB orPTB-T sequences have been altered. Such animals are useful for studyingthe function and/or activity of a PTB or PTB-T protein and foridentifying and/or evaluating modulators of PTB or PTB-T activity. Asused herein, a transgenic animal is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA whichis integrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a homologousrecombinant animal is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous PTB or PTB-T gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal may be created by introducing a PTB orPTB-T-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection or retroviral infection, and allowingthe oocyte to develop in a pseudopregnant female foster animal.Alternatively, a non-human homologue of a human PTB or PTB-T gene, suchas a rat or mouse PTB or PTB-T gene, may be used as a transgene.Intronic sequences and polyadenylation signals may also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operatively-linked to aPTB or PTB-T transgene to direct expression of a PTB or PTB-T protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866; 4,870,009; 4,873,191; and in Hogan, Manipulatingthe Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1986). Similar methods are used for production of othertransgenic animals. A transgenic founder animal may be identified basedupon the presence of a PTB or PTB-T transgene in its genome and/orexpression of PTB or PTB-T mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a PTB or PTB-T protein may further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a PTB or PTB-T gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the PTB or PTB-T gene. The PTB or PTB-T genemay be a human gene or a non-human homologue of a human PTB or PTB-Tgene. For example, a mouse PTB or PTB-T gene may be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous PTB or PTB-T gene in the mouse genome. Thehomologous recombination nucleic acid molecule may be designed suchthat, upon homologous recombination, the endogenous PTB or PTB-T gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a knock out vector). Alternatively, the homologousrecombination nucleic acid molecule may be designed such that, uponhomologous recombination, the endogenous PTB or PTB-T gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous PTB or PTB-T protein). In the homologousrecombination nucleic acid molecule, the altered portion of the PTB orPTB-T gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the PTB or PTB-T gene to allow for homologous recombinationto occur between the exogenous PTB or PTB-T gene carried by thehomologous recombination nucleic acid molecule and an endogenous PTB orPTB-T gene in a cell, e.g., an embryonic stem cell. The additionalflanking PTB or PTB-T nucleic acid sequence is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the homologous recombination nucleic acid molecule (see,e.g., Thomas and Capecchi (1987) Cell 51:503). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line, by for example electroporation, and cells inwhich the introduced PTB or PTB-T gene has homologously recombined withthe endogenous PTB or PTB-T gene are selected (see, e.g., Li, et al.(1992) Cell 69:915). The selected cells may then be injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley, In: Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, E. J. ed. (IRL, Oxford, 1987) pp.113–152). A chimeric embryo may then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellsmay be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals arewell-known (see, e.g., Bradley (1991) Current Opin. Biotechnol.2:823–829; WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

In a preferred embodiment of the invention, the stability of a CD154mRNA is regulated by modulating the level or activity of PTB or PTB-T.In another preferred embodiment, any RNA sequence operatively-linked toa cis-acting element of CD154 3′-untranslated region is regulated bymodulating the level or activity of PTB or PTB-T. RNA sequences whichmay be regulated in accordance with the invention include, but are notlimited to, viral RNA sequences, ribozymes, antisense RNA, iRNA, mRNA,rRNa, tRNA, and snRNA. It should be understood that the stability of anylength of RNA can be regulated including RNA molecules of 10 to 10000 ormore bases in length. Typically, a fusion is produced between DNAmolecules which encode the RNA sequence of interest and the cis-actingelement of CD154 3′-untranslated region. However, RNA fusions are alsocontemplated. The fusion molecule is preferably produced by standardrecombinant techniques. For example, a DNA molecule encoding the RNAsequence of interest is ligated to a DNA molecule encoding thecis-acting element and the resultant chimeric DNA molecule is expressedin a host cell to produce the fusion RNA. The DNA molecules are ligatedto each other in a 5′-to-3′ orientation such that, after ligation, theDNA molecule encoding the cis-acting element is 3′ (i.e., downstream) ofthe DNA molecule encoding the RNA sequence of interest. The fusionmolecule is than inserted into a suitable expression vector andtransformed into a suitable host cell as provided herein.

A still further aspect of the invention is a method of identifying anagent which modulates the level or activity of PTB or PTB-T. The methodprovides contacting a test cell, which contains a reporter geneoperatively-linked to a cis-acting element of a CD154 3′-untranslatedregion, with an agent and then detecting the expression of products ofnucleic acid sequences encoding the reporter in the test cell. An agentwhich causes an increase or decrease in the expression of a product ofthe nucleic acid sequence encoding the reporter in the test cell whencompared to a test cell not contacted with the agent, indicates that theagent modulates the level or activity of PTB or PTB-T in the test cell.

Test cells expressing a product of a nucleic acid sequence encoding areporter which may be used in accordance with the method of theinvention are preferably mammalian cells and most preferably humancells.

The reporter gene sequence(s) may be inserted into a recombinantexpression vector as provided herein. More than one reporter gene may beinserted into the construct such that the test cells containing theresulting construct may be assayed by different means. The test cellswhich contain the nucleic acid sequences encoding the reporter and whichexpress products of the nucleic acid sequences encoding the reporter maybe identified by at least four general approaches; detecting DNA-DNA orDNA-RNA hybridization; observing the presence or absence of marker genefunctions (e.g., resistance to antibiotics); assessing the level oftranscription as measured by the expression of reporter mRNA transcriptsin the host cell; and detecting the reporter gene product as measured byimmunoassay or by its biological activity.

The test cells may be cultured under standard conditions of temperature,incubation time, optical density, plating density and media compositioncorresponding to the nutritional and physiological requirements of thecells. However, conditions for maintenance and growth of the test cellmay be different from those for assaying candidate test compounds in thescreening methods of the invention. Modified culture conditions andmedia are used to facilitate detection of the expression of a reportermolecule. Any techniques known in the art may be applied to establishthe optimal conditions.

A reporter gene refers to any genetic sequence that is detectable anddistinguishable from other genetic sequences present in test cells.Preferably, the reporter nucleic acid sequence encodes a protein that isreadily detectable either by its presence, or by its activity thatresults in the generation of a detectable signal. A nucleic acidsequences encoding the reporter are used in the invention to monitor andreport the stability of an RNA operatively-linked to a cis-actingelement of a CD154 3′-untranslated region in test cells.

A variety of enzymes may be used as reporters including, but are notlimited to, β-galactosidase (Nolan, et al. (1988) Proc. Natl. Acad. Sci.USA 85:2603–2607), chloramphenicol acetyltransferase (CAT; Gorman, etal. (1982) Molecular Cell Biology 2:1044; Prost, et al. (1986) Gene45:107–111), β-lactamase, β-glucuronidase and alkaline phosphatase(Berger, et al. (1988) Gene 66:1–10; Cullen, et al. (1992) MethodsEnzymol. 216:362–368). Transcription of the reporter gene leads toproduction of the enzyme in test cells. The amount of enzyme present maybe measured via its enzymatic action on a substrate resulting in theformation of a detectable reaction product. The methods of the inventionprovide means for determining the amount of reaction product, whereinthe amount of reaction product generated or the remaining amount ofsubstrate is related to the amount of enzyme activity. For some enzymes,such as β-galactosidase, β-glucuronidase and β-lactamase, well-knownfluorogenic substrates are available that allow the enzyme to covertsuch substrates into detectable fluorescent products.

A variety of bioluminescent, chemiluminescent and fluorescent proteinsalso may be used as light-emitting reporters in the invention. Exemplarylight-emitting reporters, which are enzymes and require cofactor(s) toemit light, include, but are not limited to, the bacterial luciferase(luxAB gene product) of Vibrio harveyi (Karp (1989) Biochim. Biophys.Acta 1007:84–90; Stewart, et al. (1992) J. Gen. Microbiol.138:1289–1300), and the luciferase from firefly, Photinus pyralis (DeWet, et al. (1987) Mol. Cell. Biol. 7:725–737).

Another type of light-emitting reporter, which does not requiresubstrates or cofactors includes, but is not limited to, the wild-typegreen fluorescent protein (GFP) of Victoria aeguoria (Chalfie, et al.(1994) Science 263:802–805), modified GFPs (Heim, et al. (1995) Nature373:663–4; WO 96/23810), and the gene products encoded by thePhotorhabdus luminescens lux operon (luxABCDE) (Francis, et al. (2000)Infect. Immun. 68(6):3594–600). Transcription and translation of thesetype of reporter genes leads to the accumulation of the fluorescent orbioluminescent proteins in test cells, which may be measured by adevice, such as a fluorimeter, flow cytometer, or luminometer. Methodsfor performing assays on fluorescent materials are well-known in the art(e.g., Lackowicz, 1983, Principles of Fluorescence Spectroscopy, NewYork, Plenum Press).

For convenience and efficiency, enzymatic reporters and light-emittingreporters are preferred for the screening assays of the invention.Accordingly, the invention encompasses histochemical, calorimetric andfluorometric assays. An exemplary reporter construct, exemplifiedherein, contains the cis-acting element of a CD154 3′-untranslatedregion which regulates the stability of and therefore the translation(expression) of the reporter, luciferase.

Accordingly, the invention provides a method for screening for agentsthat modulate the level or activity of PTB or PTB-T comprising culturinga test cell which contains nucleic acid sequences encoding a reporteroperatively-linked to a cis-acting element of a CD154 3′-untranslatedregion; adding a test agent to a point of application, such as a well,in the plate and incubating the plate for a time sufficient to allow thetest agent to effect luciferase mRNA stability; detecting luminescenceof the test cells contacted with the test agent, wherein luminescenceindicates expression of the luciferase polypeptide in the test cells;and comparing the luminescence of test cells not contacted with the testagent. A decrease in luminescence of the test cell contacting the testagent relative to the luminescence of test cells not contacting the testagent indicates that the test agent causes a decrease in the level oractivity of PTB or an increase in the level or activity of PTB-T in thetest cell. An increase in luminescence of the test cell contacting thetest agent relative to the luminescence of test cells not contacting thetest agent indicates that the test agent causes an increase in the levelor activity of PTB or an decrease in the level or activity of PTB-T inthe test cell.

Agents which may be screened using the method provided herein encompassnumerous chemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 daltons. Agents comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Agents mayalso be found among biomolecules including peptides, antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Agents are obtained from a wide variety of sources including librariesof natural or synthetic compounds.

A variety of other reagents such as salts and neutral proteins may beincluded in the screening assays. Also, reagents that otherwise improvethe efficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, and the like may be used. The mixtureof components may be added in any order that provides for the requisitebinding.

Alternatively, antibodies against the PTB or PTB-T polypeptides mayserve as the agent to inhibit (antagonize) or stimulate (agonize) PTB orPTB-T activity. PTB or PTB-T polypeptides or epitope bearing fragmentsthereof may be used as immunogens to produce antibodies immunospecificfor such polypeptides. Various techniques well-known in the art may beused routinely to produce antibodies (Kohler and Milstein (1975) Nature256:495–497; Kozbor, et al. (1983) Immunol. Today 4:72; Cole, et al.(1985) In: Monoclonal Antibodies and Cancer Therapy, pp 77–96).

It is contemplated that agents which decrease the level or activity ofPTB or increase the level or activity of PTB-T may be used to prevent ortreat allograft rejection or inhibit CD40 activation by CD154 in asubject.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Preparation of Cytosolic and Polysomal Extracts

The Jurkat human T cell line was maintained in RPMI-1640 mediumsupplemented with 10% heat-inactivated fetal calf serum (HyClone, Logan,Utah) and 50 μg/ml gentamycin sulfate. Human PBL cells from volunteerdonors were activated with phytohemagglutinin (PHA) (Murex DiagnosticsLtd., Dartford, England) at 1 μg/ml, a concentration found to giveoptimal proliferation and lymphokine preparation. Cytoplasmicpreparations were performed using a standard method characterized forits lack of contamination by nuclear proteins (Hamilton, et al. (1993)J. Biol. Chem. 268:8881–87). Cytoplasmic lysates were prepared bywashing the cells twice in ice-cold phosphate buffered saline. Allreagents and subsequent steps were performed at 4° C. The cells werelysed by gentle resuspension in 1% TRITON® X-100 lysis buffer (50μl/2×10⁷ cells) containing 10 mM PIPES, pH 6.8, 100 mM KCl, 2.5 mMMgCl₂, 300 mM sucrose, 1 mM PEFABLOC® and 2 μg/ml each of leupeptin andpepstatin A before a 3 minute incubation followed by 3 minutecentrifugation at 500×g. The supernatant was aliquoted and stored at−80° C. as the cytoplasmic fraction. Polysomes were prepared using awell-known method (Rigby, et al. (1999) supra). Human peripheral bloodmononuclear cells from volunteer donors were homogenized in buffer A (10mM Tris-HCl, pH 7.6, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2mM DTT, 2 μg/ml leupeptin and pepstatin A, and 2 mM PEFABLOC®) andnuclei removed by centrifugation. The supernatant was layered over a 30%sucrose cushion and followed by ultracentrifugation at 36,000 rpm for 4hours at 4° C. The supernatant was removed as the S130 fraction and thepellet was resuspended in buffer A and stored in aliquots at −80° C. asthe polysome fraction.

EXAMPLE 2 RNA Binding Assays

Human CD154 3′-UTR was generated by reverse transcriptase PCR (RT-PCR)using RNA isolated from PHA-activated (16 hour) PBL, using primers thatgenerated products encoding CD154 nucleotides 12–986 and 468–835 of thehuman CD154 3′-UTR (Accession number gi 180123). Each primer set alsointroduced a SpeI restriction enzyme site at both ends. Followingamplification, the PCR products were inserted into TOPO® 2.1 vector(INVITROGEN™, Carlsbad, Calif.) and confirmed by sequencing. CD15412–986 and CD154 468–835 were excised from the TOPO® 2.1 vector withSpeI and ligated into the XbaI site of T7/T3 α-19 (GIBCO™ BRL,Gaithersburg, Md.) and confirmed by sequencing. T7/T3 CD154 483–814 delwas generated by QUICKCHANGE® (STRATAGENE®, La Jolla, Calif.) deletionfrom the T7/T3 CD154 12–986. T7/T3 CD154 12–986 was linearized with KpnIor EcoRI to generate the 12–986 and 12–292 templates, respectively.T7/T3 CD154 486–835 was linearized with EcoRI. α-³²P-labeled mRNAs withspecific activity of >108 cpm/μg RNA were prepared by in vitrotranscription by T7 RNA polymerase in the presence of 50 μCi ofα-[³²P]UTP (3000 Ci/mmole) (PERKIN-ELMER™ Life Sciences, Boston, Mass.)and 0.0125 mM UTP, and 2.5 mM ATP, GTP, and CTP (Roche Biochemicals,Indianapolis, Ind.).

RNA probes (8×10⁴ cpm; 3–14 fmole-calculated based on α-[³²P]UTPincorporation) were incubated with the specified amounts of cytoplasmicextract, nucleoplasmic extract or A₂₆₀ polysomes in 12 mM HEPES, pH 7.9,15 mM KCl, 0.2 μM dithiothreitol, 0.2 μg/ml yeast tRNA, and 10% glycerolfor 10 minutes at 30° C. UV cross-linking was performed at 4° C. usingan UV STRATALINKER® 1800 (5 minutes, 3000 microwatts/cm²) (STRATAGENE®,La Jolla, Calif.) followed by RNase digestion (10 units RNase T1 and 20μg of RNase A) for 30 minutes at 37° C. (Rigby, et al. (1999) supra).The protein-RNA complexes were separated under denaturing conditions by12% SDS-PAGE, dried, and analyzed by autoradiography. Protein-RNAcomplexes were immunoprecipitated by incubating the complexes withanti-PTB monoclonal antibody BB7 bound to protein-A SEPHAROSE® beads(Pharmacia AB, Uppsala, Sweden) for 2 hours at 4° C. Parallelimmunoprecipitation was performed with the anti-hnRNP A2 monoclonalantibody, EF67 as a specificity control (Nichols, et al. (2000) Exp.Cell Res. 256:522–532). Beads were washed six times in 100 mM NaCl,boiled in SDS-PAGE loading buffer and resolved by 12% SDS-PAGE andanalyzed by autoradiography.

Using a variety of RNA probes that varied in terms of which portion ofthe CD154 3′-UTR was contained, the binding regions for p50, p40 and p25in calf thymus, Jurkat T cells, and human PBL were identified. Thebinding of the p50, p40, and p25 RNA binding proteins in these differentextracts was mapped to nucleotides 468–835 of the human CD154 3′-UTR(SEQ ID NO:3).

EXAMPLE 3 Purification of PTB and PTB-Related Proteins

Purification of PTB and PTB-related proteins from calf thymus wasperformed as provided hereinafter and the fractions which contained p50and p25 CD154 3′-UTR RNA binding activity as measured by UV crosslinkingwere followed throughout the purification. Radiolabeled RNAcorresponding to 468–835 of the human CD154 3′-UTR was used to followthe binding activity of purified by column chromatography

Proteins were purified as follows. Fresh calf thymus (1.2 kg) wasobtained at a slaughterhouse, chopped into approximately one inch cubesand snap frozen in liquid nitrogen. The tissue was thawed overnight at4° C. in buffer A (50 mM HEPES, pH 7.5, 25 mM KCl, 5 mM MgCl₂, 250 mMsucrose, 10 mM 2-mercaptoethanol, and 1 mM PEFABLOC®). All subsequentsteps were performed at 4° C. using standard methods (Nichols, et al.(2000) supra). The tissue was ground in a blender in three liters bufferA and subsequently the crude homogenate was passed successively through2, 4, and 8 layers of cheesecloth. The suspension was centrifuged at1800×g for 7 minutes. The supernatant was transferred to clean tubes asthe cytoplasmic fraction. The nuclear pellet was resuspended in twoliters extraction buffer (250 mM sucrose, 400 mM NaCl, 50 mM HEPES pH7.5) and subsequently centrifuged at 8500 rpm in a GS3 rotor for 10minutes. The supernatant was saved as the nucleoplasmic extract andsequentially subjected to 25%, 50% and 75% ammonium sulfateprecipitation. The ammonium sulfate precipitates were resuspended indialysis buffer (50 mM NaCl, 20 mM HEPES, pH 7.5, 2 mM EDTA, 10% w/vglycerol, 10 mM 2-mercaptoethanol, and 1 mM PEFABLOC®) before 2×2 hourdialysis at 4° C.

Fractions were analyzed for the presence of CD154 3′-UTR RNA bindingproteins. The nuclear 50% ammonium sulfate fraction (120 mL) was appliedto 130 mL DEAE-SEPHACEL® (Sigma-Aldrich, St. Louis, Mo.) column. Theflow through was collected and the column washed with 500 mL of bindingbuffer (20 mM HEPES, pH 7.5, 10 mM KCl, 0.2 μM DTT, 10% glycerol, and 1mM PEFABLOC®). Proteins were eluted with 0.1, 0.3, 0.5 and 1 M KCl andanalyzed for RNA binding activity. The flow through and 0.1 M elutionfraction were combined and passed over a carboxymethyl cellulose (CMC)column and eluted with 0.1, 0.3, 0.5, and 1 M KCl step-gradient. The 0.3M elution was dialyzed against poly U SEPHAROSE® binding buffer (12 mMHEPES, pH 7.5, 15 mM KCl, 1 μg/mL yeast tRNA, 0.2 μM DTT, 100 μMPEFABLOC® and 10% glycerol) and applied to a 2 mL poly (U) Sepharose®column, washed and eluted. Specified elutions were resolved by 12%SDS-PAGE, and the p25 and p50 COOMASSIE® Blue stained bands were excisedand identified. The p50 was identified by MS/MS analysis of the trypticdigest on a Q-TOF mass spectrometer. The p25 peptide was identified byinternal amino acid sequencing of a tryptic digest.

EXAMPLE 4 Immunoblotting

Following resolution by 12% SDS-PAGE and electrotransfer tonitrocellulose, blots were blocked overnight at 25° C. in Tris-bufferedsaline/0.05% TWEEN®-20 (TBS-T) containing 3% bovine serum albumin beforeincubating 1 hour at 25° C. with a BB7 hybridoma supernatant (diluted1:2000) or affinity-purified rabbit antisera specific for the N-terminal13 amino acids of PTB (diluted 1:200) in TBS-T (1% BSA). Blots were thenwashed, incubated with 1:10000 dilution of goat-anti-mouse-HRP secondaryantibody for 1 hour at room temperature, washed five times with TBS-Tand visualized using the SUPERSIGNAL® chemiluminescence substrate(Pierce, Rockford, Ill.).

EXAMPLE 5 Cloning of the p25/PTB-T

The p25/PTB-T was cloned by RT-PCR amplification using upper and lowerprimers corresponding to the 5′-UTR and 3′-UTR of human PTB. For theupper primer, nucleotides 66–85 (5′-CCCGCGGTCTGCTCTGTGTG-3′; SEQ IDNO:7) were used, while the lower primer utilized nucleotides 1816–1839(5′-AATCTCTCGGCGGCTAGGTCACT-3′; SEQ ID NO:8). RNA from two differentdonor PHA-activated PBL as well as the Jurkat human T cell line wasisolated, reverse-transcribed with SUPERSCRIPT II™ reverse transcriptase(INVITROGEN™, Carlsbad, Calif.) using oligo-d(T) (INVITROGEN™, Carlsbad,Calif.), and PCR-amplified using Taq DNA polymerase (Roche Biochemicals,Indianapolis, Ind.). A 700 bp band was resolved by agarose gelelectrophoresis, excised, cloned into TOPO® 2.1 (INVITROGEN™, Carlsbad,Calif.), and sequenced. Identical sequences of PTB-T were seen inmultiple clones derived from each RT-PCR. PTB-T was then PCR-amplifiedfrom TOPO® 2.1/PTB-T and TA cloned into pcDNA3.1 (INVITROGEN™, Carlsbad,Calif.) and sequenced to confirm that no errors were introduced duringamplification. In vitro transcription and translation of[³⁵S]-methionine-labeled PTB-T was performed using pcDNA3.1 PTB-T vectorand PROTEINSCRIPT™ II (AMBION™, Inc., Austin, Tex.). Labeled proteinswere resolved by 12% SDS-PAGE and visualized by autoradiography. ThepTR1-Xef1α cDNA, which encodes a ˜50 kD protein, was the positivecontrol (AMBION™, Inc., Austin, Tex.).

EXAMPLE 6 Transient Transfection of Cell Lines

PTB was released from pRC/PTB (hnRNP I) vector with HindIII and ligatedinto the HindIII site in pcDNA 3.1 to yield pcDNA3.1-PTB. Luciferasereporter constructs were generated by digesting TOPO® 2.1/CD154 3′-UTR12–986 with BamHI and XhoI to release CD154 3′-UTR 104–986; ligatinginto BamHI and XhoI of pcDNA3.1. Zeo(+) (INVITROGEN™, Carlsbad, Calif.);and confirming by sequence analysis. The cDNA encoding fireflyluciferase was released from pGL3-control vector (PROMEGA®, Madison,Wis.) by XbaI and HindIII digestions, gel purified, and ligated into theBamHI site of pcDNA3.1/CD154 104–986 to yield pcDNA3.1/LUC/CD154104–986. Digestion of pcDNA3.1/LUC/CD154 104–986 with BamHI and XhoIresulted in the release of the CD154 3′-UTR; religation yieldedpcDNA3.1/LUC. The pcDNA3.1/LUC/CD154 468–835 expression plasmid wasgenerated by digesting TOPO® 2.1/CD154 468–835 with BamHI and XhoI andligating gel purified insert into the pcDNA3.1/LUC/CD154 104–986 thathad been digested with BamHI and XhoI to remove CD154 104–986. Deletionconstructs were generated by QUIKCHANGE® (STRATAGENE®, La Jolla, Calif.)deletion from TOPO® 2.1/CD154 12–986, released by BamHI/XhoI digestionand ligated into the XbaI site of pcDNA 3.1/LUC. For generation oftetracycline-repressible luciferase expression, inserts containing theCD154 3′-UTR were released by BamHI/EcoRV digestion from TOPO® vectorsprovided above and cloned into the EcoRV site downstream of theluciferase coding region in the pTRE-Luc vector (Clontech, Palo Alto,Calif.). Each vector was verified by sequencing at least twice in eachdirection.

Transient transfections were performed using 2×10⁶ Jurkat or 106 HeLacells with 0.1 μg luciferase vectors plus 6 μl LIPOFECTAMINE™ (GIBCO™BRL, Gaithersburg, Md.) in 0.5 mL RPMI for 2.5 hours at 37° C., 5% CO₂,after which 1 mL RPMI+20% FCS was added. After 20 hours, cells werelysed and luciferase activity determined using Luciferase Reporter Assay(PROMEGA®, Madison, Wis.) and luminometry. In each experiment, datarepresent the CD154 3′-UTR-specific effect by dividing the meanluciferase activity from triplicate transfections of pcDNA3.1/LUC/CD1543′UTR-containing expression plasmids by that obtained from cellstransfected with the pcDNA 3.1 LUC vector, which was assigned a value of100%. In PTB and PTB-T overexpression experiments, one μg of pcDNA3.1PTB-T, pcDNA3.1-PTB, or an empty vector control was used with 0.1 μg ofthe luciferase expression plasmids that either lacked or contained theCD154 3′-UTR and mean luciferase activity determined. In theseexperiments, the percent inhibition of CD154 3′-UTR-dependent luciferaseexpression seen with each vector was calculated and then divided by theinhibition seen with the empty control vector, which was assigned avalue of 100%.

EXAMPLE 7 Transient Transfection of Primary CD4+ T Cells

Primary human CD4 T cells (>95% purity) were isolated from PBL bynegative selection (StemCell Technologies, Vancouver, B.C.) andtransiently transfected using well-known methods (Cron, et al. (1997) J.Immunol. Methods 205:145–50). After overnight culturing with anequivalent number of irradiated (3300 rads) syngeneic whole bloodmononuclear cells in 1 μg/ml of PHA, CD4 T cells were isolated andsubjected to electroporation 19.5 hours post-PHA stimulation. Fivemillion CD4 T cells were transiently transfected with plasmid DNA in 250μl of media in 0.4 cm cuvettes at 250 V, 950 μF using a GENE PULSER®(BIO-RAD®, Hercules, Calif.). Two micrograms of eitherpcDNA3.1/LUC/CD154 104–986 or pcDNA3.1/LUC cDNA was co-transfected with2 μg of expression vector (pcDNA 3.1, pcDNA 3.1 PTB or pcDNA3.1 PTB-T)along with 1 μg of a Renilla luciferase expression control vector,pRL-null (PROMEGA®, Madison, Wis.). Cells were rested for 2 hours and 1million cells per well were stimulated in vitro with PMA (25 ng/ml) andionomycin (1.5 μM) or media alone for 6 hours at 37 C. Cells were washedand lysed, and luciferase activity was determined using a DualLuciferase assay kit (PROMEGA®, Madison, Wis.) and a LB9507 luminometer(EG&G Wallac, Bad Wildbad, Germany). Data was analyzed in duplicate andcorrected for transfection efficiency based on Renilla luminescence(Cron, et al. (2000) Clin. Immunol. 94:179–91).

EXAMPLE 8 Northern/RT-PCR Light Cycler RNA Analysis

Jurkat cells were transiently transfected as described and totalcellular RNA was extracted by acid guanidinium-phenol-chloroformextraction (Chomczynski and Sacchi (1987) Anal. Biochem. 162:156–159)modified by increasing the 2-mercaptoethanol (Sigma-Aldrich, St Louis,Mo.) from 0.1 M to 0.7 M in the 5 M guanidinium thiocyanate (Fluka,Switzerland) denaturing solution. RNA was size fractionated byformaldehyde-agarose gel electrophoresis and blotted to HYBOND™-N nylonmembrane (Amersham Corp., Arlington Heights, Ill.) in 20×SSC, and bakedunder vacuum at 80° C. for 2 hours. The northern blot was sequentiallyhybridized with an end-labeled luciferase primer(5′-GGTACTTCGTCCACAAACACAACTCC-3′; SEQ ID NO:9) and oligo-labeled HLA-B7cDNA and visualized by autoradiography, with quantification performed byphosphorimaging using the PHOSPHORIMAGER™ 445 SI (MOLECULAR DYNAMICS™,Sunnyvale, Calif.). A separate transfection was analyzed by real timePCR. Total cellular RNA was extracted using RNEASY® Kit (QIAGEN®,Valencia, Calif.) and poly(A)+ RNA isolated using OLIGOTEX® beads(QIAGEN®, Valencia, Calif.). Poly(A)+ RNA was digested with DNase I(AMBION®, Inc., Austin, Tex.) prior to reverse transcribing with oligodT and SUPERSCRIPT II™ RT (INVITROGEN™, Carlsbad, Calif.). Reversetranscriptions were analyzed for luciferase transcripts using5′-GGTGGCTCCCGCTGAATTGG-3′ (SEQ ID NO:10) (upper primer) and5′-CCGTCATCGTCTTTCCGTGC-3′ (SEQ ID NO:11) (lower primer) and SYBER®Green PCR Core Reagents (APPLIED BIOSYSTEMS™, Foster City, Calif.) byreal time PCR using an ICYCLERT™ (BIO-RAD®, Hercules, Calif.). Each RTreaction was simultaneously examined for GAPDH transcript as a control.The luciferase/GAPDH transcript ratio was calculated for each sample,based on the manufacturer's instructions. For studies of mRNA stability,TET-OFF™ HeLa cells (Clontech, Palo Alto, Calif.) were obtained and usedaccording to manufacturer's instructions. Transient transfection ofthese cell was as described above. Cells were allowed to recoverovernight, then treated with Doxycyline (1 μg/ml) to shut offtranscription for specified times. RNA extraction and analysis wasperformed as described above.

1. A method for identifying agents that modulate the level or activityof a polypyrimidine tract protein comprising contacting a test cell,which contains a polypyrimidine tract protein and a cis-acting elementof a CD154 3′-untranslated region operatively-linked to a nucleic acidsequence encoding a reporter, with an agent and detecting the expressionof a product of the nucleic acid sequence encoding the reporter in thetest cell.
 2. The method of claim 1, wherein a decrease in theexpression of a product of the nucleic acid sequence encoding thereporter, in the test cell contacted with the agent relative to theexpression of the product of the nucleic acid sequence encoding thereporter in a test cell not contacted with the agent, indicates that theagent causes a decrease in the level or activity of polypyrimidine tractprotein of SEQ ID NO:2 or an increase in the level or activity ofpolypyrimidine tract protein isoform of SEQ ID NO:1.
 3. The method ofclaim 1, wherein an increase in the expression of a product of thenucleic acid sequence encoding the reporter in the test cell contactedwith the agent relative to the expression of the product of the nucleicacid sequence encoding the reporter in a test cell not contacted withthe agent, indicates that the agent causes an increase in the level oractivity of polypyrimidine tract protein of SEQ ID NO:2 or a decrease inthe level or activity of polypyrimidine tract protein isoform of SEQ IDNO:1.